Dear all,
I'm currently analysing RNAseq data (Illumina paired end, read length 101 bases) in order to investigate different cancer stages.
So far, I ran the Tuxedo pipeline (Tophat + Cufflinks) in two different versions and my colleaque ran CLC Bio in the most recent version in order to compare the results.
What bothers me is:
performing the whole Tuxedo pipeline twice and then analysing the results using cummeRbund (as described in the workflow published in NBT), there are NO genes found to be significantly different between two distinct conditions.
However, CLC Bio nicely finds genes that are differentially expressed and one of them could already be validated in the lab.
I now switched to GSNAP for the alignments, hoping to get better results, still this cannot be the main reason for these differences.
So my question is: what am I doing wrong? My best guess is that the Cufflinks steps are somehow not working properly, but since everything runs through without any error messages, I don't really have a starting point for backtracking mistakes.
Any help is greatly appreciated!
Best regards
M&M:
Alignments were performed with the stable version against Hg18 using the following commands:
and against Hg19 with the beta versions of Tophat (2.0.1), bowtie beta 5 and Cufflinks 2.0.1 using the following commands:
I'm currently analysing RNAseq data (Illumina paired end, read length 101 bases) in order to investigate different cancer stages.
So far, I ran the Tuxedo pipeline (Tophat + Cufflinks) in two different versions and my colleaque ran CLC Bio in the most recent version in order to compare the results.
What bothers me is:
performing the whole Tuxedo pipeline twice and then analysing the results using cummeRbund (as described in the workflow published in NBT), there are NO genes found to be significantly different between two distinct conditions.
However, CLC Bio nicely finds genes that are differentially expressed and one of them could already be validated in the lab.
I now switched to GSNAP for the alignments, hoping to get better results, still this cannot be the main reason for these differences.
So my question is: what am I doing wrong? My best guess is that the Cufflinks steps are somehow not working properly, but since everything runs through without any error messages, I don't really have a starting point for backtracking mistakes.
Any help is greatly appreciated!
Best regards
M&M:
Alignments were performed with the stable version against Hg18 using the following commands:
Code:
tophat -p 8 -o mapping1 bowtie-0.12.7/genomes/hg18/hg18 R1_001.fastq R2_001.fastq cufflinks --GTF bowtie-0.12.7/genomes/hg18/hg18_annotations.gtf -p 8 -o annotation1 mapping1/accepted_hits.bam cuffmerge -g bowtie-0.12.7/genomes/hg18/hg18_annotations.gtf -s bowtie-0.12.7/genomes/hg18/hg18.fa -p 8 assemblies.txt cuffdiff -m 199 -s 38 -o diff_out -b bowtie-0.12.7/genomes/hg18/hg18.fa -p 8 -L G1,G2 -u merged_asm/merged.gtf mapping1/accepted_hits.bam,mapping2/accepted_hits.bam,mapping3/accepted_hits.bam mapping4/accepted_hits.bam,mapping5/accepted_hits.bam,mapping6/accepted_hits.bam
and against Hg19 with the beta versions of Tophat (2.0.1), bowtie beta 5 and Cufflinks 2.0.1 using the following commands:
Code:
tophat --b2-sensitive -p 8 -o mapping1 bowtie2-2.0.0-beta6/hg19/hg19 R1_001.fastq R2_001.fastq cufflinks --GTF UCSC_annotation_hg19.gtf -p 8 -o annotation1 mapping1/accepted_hits.bam cuffmerge -g UCSC_annotation_hg19.gtf -s bowtie2-2.0.0-beta6/hg19/hg19.fa -p 8 assemblies.txt cuffdiff -m 199 -s 38 -o diff_out -b bowtie2-2.0.0-beta6/hg19/hg19.fa -p 9 -L G1,G2 -u merged_asm/merged.gtf mapping1/accepted_hits.bam,mapping2/accepted_hits.bam,mapping3/accepted_hits.bam mapping4/accepted_hits.bam,mapping5/accepted_hits.bam,mapping6/accepted_hits.bam
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